Summary. A cell line designated SKM‐1 was newly established from leukaemic cells of a 76‐year‐old Japanese male patient with monoblastic leukaemia following myelodysplastic syndrome (MDS). The cells were obtained from peripheral blood of the patient when he lost multiple point mutations of ras genes with acquisition of chromosomal abnormalities during disease progression in MDS. The cells grew as a single floating cell, and have been continuously growing with the morphological characteristics of immature monoblasts by serial passages during the past 42 months with a doubling time of about 48 h. By cytochemical analysis. the cloned cells were positive for butyrate esterase, but negative for the Epstein‐Barr virus associated nuclear antigen. Phenotypic analysis revealed the expression of myelomonocyte specific antigens such as CD4, CD13, CD33 and HLA‐DR. Cells from the primary peripheral blood and those from SO passages of the SKM‐1 cell line both possessed no activated ras genes but showed karyotype abnormalities with 46.XY, del(9)(q13;q22), der(17) t(17:?)(p13:?). The SKM‐1 cells have two mutations in p53 gene and overexpress the pS3 products. This cell line may contribute to a better understanding of molecular mechanisms in the progression from MDS to myelogenous leukaemia.

A novel cell line SKNO-1 was established from the bone marrow cells of a 22-year-old male suffering from acute myeloblastic leukaemia (AML) M2 with t(8;21) whose disease became resistant to chemotherapy after acquisition of 1 7 monosomy. SKNO-1 has been maintained for more than 36 months as a granulocyte-macrophage colonystimulating factor (GM-CSF) dependent line.Morphologically, SKNO-1 cells were myeloblasts somewhat maturated. The cells grow in suspension with a doubling time of 48-72h. The survival and growth of SKNO-1 cells was absolutely dependent on granulocytemacrophage colony stimulating factor (GM-CSF). SKNO-1 cells possessed t(8;21) and monosomy 1 7 which were observed in original leukaemic cells. We confirmed that the AMLl gene, located on chromosome 21, was rearranged and the AML1-MTG8 fusion transcript was expressed in SKNO-1 cells. Over-expression and mutation of the p53 gene were also detected in SKNO-1. It is likely that alterations of AMLl or MTG8 gene and p53 gene contribute to a disease progression in this case.Since t(8;2 1) translocation is a common chromosornc abnormality in AML, and inactivation of the p53 gene may play a crucial role in disease progression in AML, SKNO-1 would be a useful tool for analysing the molccular mechanisms in myeloid leukaemogenesis.

We analyzed activating mutations of N-ras and K-ras by the polymerase chain reaction and oligonucleotide hybridization in hematological disorders. Activating mutations of these codons were detected in 4 of 20 cases of myelodysplastic syndrome (MDS) and 15 of 77 cases of acute myelogenous leukemia (AML). Out of 19 cases of MDS and AML who carried active mutations, 7 cases were found to have two or more distinct mutations in activating codons of N-ras and K-ras. Ras mutation was found preferentially in progressive disease such as refractory anemia with excess of blasts (RAEB) of RAEB in transformation (RAEB-t). A relatively high incidence of ras mutation was found in M₅ AML (40%). No ras mutations were found in other hematological disorders, such as acute lymphoblastic leukemia and chronic myelogenous-leukemia. The most frequent amino acid substitution was that of an aspartate for glycine at codon 12 of N-ras resulting from G to A mutation (11/35). The survival of AML patients who carried ras mutations showed no significant differences from those without ras mutations calculated by Kaplan-Meier. Seven cases of MDS and 7 cases of AML patients could be investigated at various points during their clinical course. Among these 14 cases, we found 2 interesting cases of MDS. The first case lost multiple clones carrying ras mutations during disease progression, the second case acquired mutation of the ras gene during disease progression. These results suggested that multiple point mutations of ras genes may not be initiating events but may contribute to a clonal evolution of MDS and AML.

Although molecular and cytogenetic studies strongly point to the role of oncogenes, the mechanisms underlying the development of MDS and their progressive evolution to AML are still largely unknown. It has been postulated that AML has a preleukemic stage and a multi step pathogenesis, with the preleukemic stem cell able to undergo clonal evolution, with the acquisition of karyotypic abnormalities, leading to the development of acute leukemic subclones. The activations of the ras oncogenes or inactivation of the p53 anti-oncogene by point mutations have been described recently in several cases of MDS as well as AML, suggesting a critical role for these alterations in the development of these myelogenous leukemias. We reported previously establishment of a leukemic cell line, SKM-1, from the patient who initially possessed multiple point mutations of ras genes but lost these mutations during disease progression to myelomonocytic leukemia with acquisition of chromosomal abnormalities involving the p53 anti-oncogene. This process is characterized by genetic instabilities probably due to the failure of their DNA repairment leading to abnormal control of cell proliferation and differentiation. Studying this cell line, SKM-1, is a promising approach to understand the mechanisms of the initiation, disease progression, alterations of DNA repairment, and genetic instability in MDS and myelogenous malignancies.

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a lymphoma-like disease. Although the histologic appearance of this disease is benign, it is clinically malignant, with chemotherapy being ineffective and the prognosis being poor. In this article, the authors report the results of treating two patients with AILD with cyclosporin A. One patient had cervical lymphadenopathy and fever, and AILD was refractory to both chemotherapy and a-interferon. However, treatment with cyclosporin A achieved complete remission. The other patient had generalized lymphadenopathy, and cyclosporin A was used as the treatment of first choice. Complete remission also was achieved in this patient, who still is in remission. These two cases demonstrate that cyclosporin A can be effective for treating AILD.